Abstact

Polyethylene terephthalate (PET) plastic waste causes serious environmental pollution due to insufficient recycling rates. Enzymatic PET depolymerization offers a sustainable recycling strategy, but limited stability and activity of current PET-degrading enzymes restrict practical implementation. Here, we engineer Polyester Hydrolase Leipzig 7 (PHL7), a PET hydrolase from a compost metagenome, to enhance its stability and catalytic performance under recycling-relevant conditions. Using Rosetta PROSS-based computational design combined with rational mutagenesis, we introduce up to 24 mutations, generating variants with melting temperatures of 88-95 degrees C and over 110-fold higher activity in 0.1 M phosphate buffer compared to the parent enzyme. Benchmarking shows that the best variants (R4M6, R4M9, and R4M10) match or exceed the performance of established engineered PET hydrolases, including ICCG and LCC-A2, and approach that of TurboPETase across multiple conditions. Under high substrate loadings, the PHL7-R4 variants degrade 75-78% of 10% (w/w) PET within 24 h at 65 degrees C, outperforming ICCG, while an optimized variant R4M10-H185Y achieves up to 84% degradation of 20% (w/w) PET. X-ray structure determination and molecular dynamics simulations reveal key stabilizing and activity enhancing mechanisms. These engineered PHL7 variants represent robust biocatalysts for scalable enzymatic PET recycling.